Identification of new genes of nodule bacteria sinorhizobium meliloti involved in the control of efficiency of symbiosis with alfalfa medicago sativa

Cover Page

Cite item

Full Text

Abstract

Background. Alfalfa root nodule bacteria (Sinorhizobium meliloti) are among the most active symbiotic N2-fixers. Their symbiotic efficiency (SE) defined as an ability to enhance the productivity of inoculated host plants is the polygenic trait controlled by a complicated system of genes, inactivation of which can result in either decrease or increase of SE. Analysis of previously identified eff-genes, whose mutations result in SE increase, revealed their location in different parts of genome (chromosome or megaplasmids) and demonstrated that these genes are not involved in operation of nitrogenase system. Mutations in these genes have pleiotropic effects, changing various cultural-biochemical properties of bacteria. Materials and methods. The object of research were the laboratory S. meliloti strain CXM1-105 and its Tn5-mutants with Eff++ phenotype, which are able to grow in diagnostic media containing indicator of cell redox potential ТТC, herbicide 2М-4ХМ or the Congo Red dye. New eff-genes were identified using the modified method of “inverted” PCR. Results. We obtained three Tn5 mutants with an increased SE in which the symbiotic phenotypes are dependent on the host. Two-factor analysis of variance demonstrated that the genotypic difference between mutants is most pronounced under the salt stress, while in its absence SE is determined mostly by the host genotype. Molecular-biological analysis revealed that the T4 mutant harbours the Tn5 insertion in the mtaD gene, T795 - in the thiC gene, and M3 - in the gene which encodes a protein belonging to the GntR family of transcription regulators. Conclusion. We demonstrated firstly that mutations in genes involved in transcription regulation, phosphonate metabolism and thiamine biosynthesis may result in a SE increase. The “inverted” PCR method enabled us to rapidly extract DNA fragments flanking the transposon, which suggests applicability of this method for identification of new rhizobia genes marked by Tn5.

About the authors

Olga Petrovna Onishchuk

All-Russia Research Institute for Agricultural Microbiology

Email: olony@yandex.ru
PhD, Senior scientists of the laboratory of genetic and selection of microorganisms

Oksana Nikolayevna Kurchak

All-Russia Research Institute for Agricultural Microbiology

Email: genet@yandex.ru
PhD, Senior scientists of the laboratory of genetic and selection of microorganisms

Elena Petrovna Chizhevskaya

All-Russia Research Institute for Agricultural Microbiology

Email: genet@yandex.ru
PhD, Senior scientists of the laboratory of genetic and selection of microorganisms

Evgeniy Yevgenyevich Andronov

All-Russia Research Institute for Agricultural Microbiology

Email: eeandr@yandex.ru
PhD, Head of the laboratory of microbiological monitoring and bioremediation of soils

Boris Vasilyevich Simarov

All-Russia Research Institute for Agricultural Microbiology

Email: genet@yandex.ru
Dr. Sci., Head of the laboratory of genetic and selection of microorganisms

References

  1. Ибрагимова М. В., Румянцева М. Л., Онищук О. П. с соавт. (2006). Симбиоз клубеньковых бактерий Sinorhizobium meliloti с люцерной Medicago sativa в условиях засоления. Микробиология. Т. 75 (1): С. 94-100.
  2. Кожемяков А. П., Тихонович И. А. (1998). Использование инокулянтов бобовых и биопрепаратов комплексного действия в сельском хозяйстве. Докл. РАСХН. № 6: С. 7-10.
  3. Лакин Г. Ф. (1990). Биометрия (4-е издание). М.: Высш. школа.
  4. Маниатис Т., Фрич Э., Сэмбрук Дж. (1984). Методы генной инженерии. Пер. с англ. М.: Мир.
  5. Онищук О. П., Курчак О. Н., Шарыпова Л. А. c соавт. (2001). Анализ различных типов конкурентоспособности у Tn5-мутантов клубеньковых бактерий люцерны (Sinorhizobium meliloti). Генетика. Т. 37 (9): С. 1-6.
  6. Онищук О. П., Шарыпова Л. А., Курчак О. Н. c соавт. (2005). Выявление генов Sinorhizobium meliloti, влияющих на синтез поверхностных полисахаридов и конкурентоспособность. Генетика. Т. 41 (12): С. 1617-1623.
  7. Проворов Н. А., Воробьев Н. И. (2012). Генетические основы эволюции растительно-микробного симбиоза. Под ред. И. А. Тихонович. СПб.: Информ-Навигатор.
  8. Проворов Н. А., Жуков В. А., Курчак О. Н. с соавт. (2013). Совместная миграция клубеньковых бактерий и бобовых растений в новые местообитания: механизмы коэволюции и практическое значение (обзор). Прикладная биохимия и микробиология. Т. 49 (3): С. 229-235.
  9. Федоров С. Н., Симаров Б. В. (1987). Получение мутантов с измененными симбиотическими свойствами у Rhizobium meliloti под действием УФ-лучей. С.-х. биология. № 9: С. 44-49.
  10. Чижевская Е. П., Кроль Е. А., Онищук О. П. с соавт. (1998). Физическое и генетическое картирование мутаций симбиотической эффективности на мегаплазмиде-2 штамма СХМ1 Rhizobium meliloti. Генетика. Т. 34 (9): С. 1220-1227.
  11. Чижевская Е. П., Онищук О. П., Андронов Е. Е., Симаров Б. В. (2011). Использование метода сайт-направленного мутагенеза для изучения функций гена SMb20332 у клубеньковых бактерий Sinorhizobium meliloti. С.-х. биология. № 3: С. 55-60.
  12. Юргель С. Н., Шарыпова Л. А., Симаров Б. В. (1998) Tn5-мутации Rhizobium meliloti, вызывающие повышение редокс-потенциала свободноживущих клеток и эффективности их симбиоза с люцерной. Генетика. Т. 34 (6): С. 737-741.
  13. Barnet M. J., Fisher R. F., Jones T. et al. (2001). Nucleotide sequence and predicted functions of the entire Sinorhizobium meliloti pSymA megaplasmid. Proc. Natl. Acad. Sci. U. S.A. V. 98 (17): P. 9883-9888.
  14. Beringer J. E. (1974). R1 transfer in Rhizobium leguminosarum. J. Gen. Microbiol. V. 84: P. 188-198.
  15. Capela D., Barloy-Hubler F., Gouzy J. et al. (2001). Analysis of the chromosome sequence of the legume symbiont Sinorhizobium meliloti strain 1021. Proc. Natl. Acad. Sci. U. S. A. V. 98 (17): P. 9877-9882.
  16. Finan T. M., Kunkel B., De Vos G. F. et al. (1986). Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes. J. Bacteriol. V. 167 (1): P.66-72.
  17. Finan T. M., Weidner S., Wong K. et al. (2001). The complete sequence of the 1,638-kb pSymB megaplasmid from the N2-fixing endosymbiont Sinorhizobium meliloti. Proc. Natl. Acad. Sci. U. S.A. V. 98 (17): P. 9889-9894.
  18. Meade H. M., Long S. R., Ruvkun G. B. et al. (1982). Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon mutagenesis. J. Bacteriol. V. 149 (1): P. 114-122.
  19. Olah B., Kiss E., Györgypal Z. et al. (2001). Mutation in the ntr gene, a member of the vap gene family, increase the symbiotic efficiency of Sinorhizobium meliloti. MPMI. V. 14 (7): P. 887-894.
  20. Pobigailo N., Szymczak S., Nattkemper T. W., Becker A. (2008). Identification of genes relevant to symbiosis and competitiveness in Sinorhizobium meliloti using signature-tagged mutants. MPMI. V. 21 (2): P. 219-231.
  21. Sharypova L. A., Onischchuk O. P., Chesnokova O. N., et al. (1994). Isolation and characterization of Rhizobium meliloti Tn5 mutants showing enhanced symbiotic effectiveness. Microbiology. V. 140: P. 463-470.
  22. Sharypova L. A., Pretorius-Guth I.-M., Simarov B. V., Puhler A. (1992). Genetic improvement of Rhizobium strains. In: G. F. Hong (Ed.). The nitrogen fixation and its research in China. Berlin: Springer-Verlag. P. 266-285.
  23. Sharypova L. A., Simarov B. V. (1995). Identification of genes affecting symbiotic effectiveness of Rhizobium meliloti. In: I. A. Tichonovich et al. (Eds.). Nitrogen fixation: Fundamental and applications. Dordrecht: Kluwer Acad. Publ. P. 371-376.
  24. Sharypova L. A., Yurgel S. N., Keller M. et al. (1999). The eff-482 locus of Sinorhizobium meliloti CXM1-105 that influences symbiotic effectiveness consists of three genes encoding an endoglucanase, a transcriptional regulator and an adenylate cyclase. Molec.Gen.Genet. V. 261: P. 1032-1044.
  25. Simon R., Priefer U., Pühler A. (1983) Vector plasmids for in vitro manipulations of gram-negative bacteria. In: A. Pühler, editor. Molecular genetics of the bacteria-plant interaction. Springer-Verlag. Berlin. Heidelberg: P. 98-106.

Copyright (c) 2014 Onishchuk O.P., Kurchak O.N., Chizhevskaya E.P., Andronov E.Y., Simarov B.V.

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.
 


This website uses cookies

You consent to our cookies if you continue to use our website.

About Cookies